The mammalian genome is not merely a static combination of the four genetic codes A, T, C, and G. Reversible chemical modifications on DNA and histones contribute significantly to cell diversity through dynamic regulation of global gene expression. Prior to our work, no example of reversible chemical modifications on RNA that could affect gene expression had been shown. In the last funding period, we discovered the first two RNA demethylases: FTO, a protein associated with human fat mass obesity, and ALKBH5, a protein that affects spermatogenesis in a mouse model. These two proteins belong to the AlkB family iron- and 2-ketoglutarate (2- KG)-dependent dioxygenases and catalyze oxidative demethylation of the most prevalent internal modifications of mammalian messenger RNA (mRNA) and other nuclear RNA, N6- methyladenosine (m6A). These studies provided the first demonstration of reversible RNA modification that may impact biological regulation analogous to the well-known reversible DNA and histone chemical modifications. We have also discovered proteins that can selectively recognize m6A-modified mRNA. We have confirmed that binding of m6A-containing mRNA by a family of the reader proteins affects the translation status and lifetime of the target mRNA. In the current application, we plan to thoroughly characterize the structures of FTO with bound nucleic acid substrates. With the structural information available, we plan to apply photo-crosslinking as well as m6A-seq to mammalian cells in order to capture and identify RNA substrates of FTO. With substrates identified and potential partner proteins revealed, we hope to investigate and elucidate the mechanisms of the demethylation-based regulatory process. In addition to mRNA m6A demethylation, we have recently discovered that certain transfer RNA (tRNA) modification is also reversible. A primary function of tRNA modifications is commonly thought to control the quality and quantity of protein synthesis. We found that the human ALKBH1 is a tRNA demethylase acting on N1-methyladenosine (m1A) residues, which are known to control the stability and folding of many tRNA species. This discovery provides another example of reversible methylation in a different RNA species, indicating that RNA demodification is a general regulatory mechanism in biology. Furthermore, it raises many new questions on how reversible tRNA methylation impacts tRNA biogenesis and regulates translation. We plan to thoroughly establish the functional roles and elucidate the underlying mechanisms of this new mode of tRNA methylation/demethylation in biological regulation.